2-Aminopyridine and 2-pyridone C-nucleosides, oligonucleotides comprising, and tests using the same oligonucleotides

ABSTRACT

The present invention provides 2-aminopyridine and 2-pyridone C-nucleosides and oligonucleotides containing the subject nucleosides. The nucleosides are useful in the preparation of the subject oligonucleotides. The oligonucleotides are useful in oligonucleotide-based diagnosis and separation through triplex binding.

This Application claims benefit of Provisional No. 60/023,241 filed Aug.9, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to enhanced triplex binding andcompositions useful in achieving enhanced triplex binding. In someembodiments, the invention provides modified nucleosides that provideenhanced triplex binding of single stranded oligonucleotides containingone or more of the modified nucleosides to target duplexoligonucleotides. Also provided by the invention are methods of usingthe modified nucleosides to prepare oligonucleotides containing them andmethods of using the oligonucleotides containing the modifiednucleosides in triplex binding assays and methods of separation.

2. Brief Description of Related Art

Hsieh, H.-P. and McLaughlin, L. W., J. Org. Chem. (1995), 60:5356-5359,discloses the syntheses of two pyridine C-nucleosides as“deletion-modified” analogues of dT and dC.

OBJECTS OF THE INVENTION

Selected embodiments of the present invention accomplish one or more ofthe following objects:

A principal object of the invention is sequence specific triplex bindingof single stranded oligonucleotides to duplex oligonucleotides. Inparticular an object is diagnosis through triplex binding.

An additional object of the invention is to provide 2-aminopyridine and2-pyridone C-nucleosides.

An additional object of the invention is to provide oligonucleotidescontaining 2-aminopyridine and 2-pyridone C-nucleosides.

An additional object of the invention is to provide 2-aminopyridine and2-pyridone C-nucleosides as intermediates in the preparation ofoligonucleotides containing 2-aminopyridine and 2-pyridoneC-nucleosides.

SUMMARY OF THE INVENTION

Compounds or compositions having formula (I) or (II) are providedherein:

wherein:

each R¹ is independently H or a hydroxy protecting group, or both R¹groups are taken together to form a cyclic hydroxy protecting group;

R² is H, F, —OR¹, or —OR⁶;

R³ is H or —CH₃;

each R⁴ of formula I and II is independently H or an amine protectinggroup, or both R⁴ groups of formula I are taken together to form acyclic amine protecting group;

R⁵ is H^(, —CH) ₃ or —C≡C—CH₃; and

R⁶ is

and salts, solvates, resolved enantiomers and purified diastereomersthereof.

Another aspect of the invention is directed to oligomers capable oftriple helix formation comprising a multiplicity of nucleosides linkedby internucleoside linkages wherein at least one nucleoside is amodified nucleoside comprising a nucleoside composition of theinvention.

Another aspect of the invention is directed to methods of detecting thepresence, absence or amount of a particular DNA duplex in a samplesuspected of containing DNA comprising contacting the sample with anoligomer of the invention under conditions wherein a triple helix isformed between the oligomer and the particular DNA duplex.

DETAILED DESCRIPTION

The present invention is directed to 2-aminopyridine and 2-pyridoneC-nucleosides. In particular the invention is directed to compositionscomprising a compound of the formula:

Each R¹ is independently H or a hydroxy protecting group, or both R¹groups are taken together to form a cyclic hydroxy protecting group.Typically, each R¹ is H.

Typical R¹ hydroxy protecting groups described in Greene (pages 14-118)include Ethers (Methyl); Substituted Methyl Ethers (Methoxymethyl,Methylthiomethyl, t-Butylthiomethyl, (Phenyldimethylsilyl)methoxymethyl,Benzyloxymethyl, p-Methoxybenzyloxymethyl, (4-Methoxyphenoxy)methyl,Guaiacolmethyl, t-Butoxymethyl, 4-Pentenyloxymethyl, Siloxymethyl,2-Methoxyethoxymethyl, 2,2,2-Trichloroethoxymethyl,Bis(2-chloroethoxy)methyl, 2-(Trimethylsilyl)ethoxymethyl,Tetrahydropyranyl, 3-Bromotetrahydropyranyl, Tetrahydropthiopyranyl,1-Methoxycyclohexyl, 4-Methoxytetrahydropyranyl,4-Methoxytetrahydrothiopyranyl, 4-MethoxytetrahydropthiopyranylS,S-Dioxido, 1-[(2-Chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl, 35,1,4-Dioxan-2-yl, Tetrahydrofuranyl, Tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-Octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl));Substituted Ethyl Ethers (1-Ethoxyethyl, 1-(2-Chloroethoxy)ethyl,1-Methyl-1-methoxyethyl, 1-Methyl-1-benzyloxyethyl,1-Methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-Trichloroethyl,2-Trimethylsilylethyl, 2-(Phenylselenyl)ethyl, t-Butyl, Allyl,p-Chlorophenyl, p-Methoxyphenyl, 2,4-Dinitrophenyl, Benzyl); SubstitutedBenzyl Ethers (p-Methoxybenzyl, 3,4-Dimethoxybenzyl, o-Nitrobenzyl,p-Nitrobenzyl, p-Halobenzyl, 2,6-Dichlorobenzyl, p-Cyanobenzyl,p-Phenylbenzyl, 2- and 4-Picolyl, 3-Methyl-2-picolyl N-Oxido,Diphenylmethyl, p,p′-Dinitrobenzhydryl, 5-Dibenzosuberyl,Triphenylmethyl, α-Naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, Di(p-methoxyphenyl)phenylmethyl,Tri(p-methoxyphenyl)methyl, 4-(4′-Bromophenacyloxy)phenyldiphenylmethyl,4,4′,4″-Tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-Tris(levulinoyloxyphenyl)methyl,4,4′,4″-Tris(benzoyloxyphenyl)methyl,3-(Imidazol-1-ylmethyl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-Bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-Anthryl,9-(9-Phenyl)xanthenyl, 9-(9-Phenyl-10-oxo)anthryl,1,3-Benzodithiolan-2-yl, Benzisothiazolyl S,S-Dioxido); Silyl Ethers(Trimethylsilyl, Triethylsilyl, Triisopropylsilyl,Dimethylisopropylsilyl, Diethylisopropylsily, Dimethylthexylsilyl,t-Butyldimethylsilyl, t-Butyldiphenylsilyl, Tribenzylsilyl,Tri-p-xylylsilyl, Triphenylsilyl, Diphenylmethylsilyl,t-Butylmethoxyphenylsilyl); Esters (Formate, Benzoylformate, Acetate,Choroacetate, Dichloroacetate, Trichloroacetate, Trifluoroacetate,Methoxyacetate, Triphenylmethoxyacetate, Phenoxyacetate,p-Chlorophenoxyacetate, p-poly-Phenylacetate, 3-Phenylpropionate,4-Oxopentanoate (Levulinate), 4,4-(Ethylenedithio)pentanoate, Pivaloate,Adamantoate, Crotonate, 4-Methoxycrotonate, Benzoate, p-Phenylbenzoate,2,4,6-Trimethylbenzoate (Mesitoate)); Carbonates (Methyl,9-Fluorenylmethyl, Ethyl, 2,2,2-Trichloroethyl, 2-(Trimethylsilyl)ethyl,2-(Phenylsulfonyl)ethyl, 2-(Triphenylphosphonio)ethyl, Isobutyl, Vinyl,Allyl, p-Nitrophenyl, Benzyl, p-Methoxybenzyl, 3,4-Dimethoxybenzyl,o-Nitrobenzyl, p-Nitrobenzyl, S-Benzyl Thiocarbonate,4-Ethoxy-1-naphthyl, Methyl Dithiocarbonate); Groups With AssistedCleavage (2-Iodobenzoate, 4-Azidobutyrate, 4-Niotro-4-methylpentanoate,o-(Dibromomethyl)benzoate, 2-Formylbenzenesulfonate,2-(Methylthiomethoxy)ethyl Carbonate, 4-(Methylthiomethoxy)butyrate,2-(Methylthiomethoxymethyl)benzoate); Miscellaneous Esters(2,6-Dichloro-4-methylphenoxyacetate, 2,6-Dichloro-4-(1,1,3,3tetramethylbutyl)phenoxyacetate,2,4-Bis(1,1-dimethylpropyl)phenoxyacetate, Chorodiphenylacetate,Isobutyrate, Monosuccinoate, (E)-2-Methyl-2-butenoate (Tigloate),o-(Methoxycarbonyl)benzoate, p-poly-Benzoate, α-Naphthoate, Nitrate,Alkyl N,N,N′,N′-Tetramethylphosphorodiamidate, N-Phenylcarbamate,Borate, Dimethylphosphinothioyl, 2,4-Dinitrophenylsulfenate); andSulfonates (Sulfate, Methanesulfonate (Mesylate), Benzylsulfonate,Tosylate).

More typically, R¹ hydroxy protecting groups include substituted methylethers, substituted benzyl ethers, silyl ethers, and esters includingsulfonic acid esters, still more typically, trialkylsilyl ethers,tosylates and acetates.

Typical 1,2-diol protecting groups (thus, generally where two OH groupsare taken together with the R¹ protecting functionality) are describedin Greene at pages 118-142 and include Cyclic Acetals and Ketals(Methylene, Ethylidene, 1-t-Butylethylidene, 1-Phenylethylidene,(4-Methoxyphenyl)ethylidene, 2,2,2-Trichloroethylidene, Acetonide(Isopropylidene), Cyclopentylidene, Cyclohexylidene, Cycloheptylidene,Benzylidene, p-Methoxybenzylidene, 2,4-Dimethoxybenzylidene,3,4-Dimethoxybenzylidene, 2-Nitrobenzylidene); Cyclic Ortho Esters(Methoxymethylene, Ethoxymethylene, Dimethoxymethylene,1-Methoxyethylidene, 1-Ethoxyethylidine, 1,2-Dimethoxyethylidene,α-Methoxybenzylidene, 1-(N,N-Dimethylamino)ethylidene Derivative,α-(N,N-Dimethylamino)benzylidene Derivative, 2-Oxacyclopentylidene);Silyl Derivatives (Di-t-butylsilylene Group,1,3-(1,1,3,3-Tetraisopropyldisiloxanylidene), andTetra-t-butoxydisiloxane-1,3-diylidene), Cyclic Carbonates, CyclicBoronates, Ethyl Boronate and Phenyl Boronate.

More typically, 1,2-diol protecting groups include those shown in TableA, still more typically, epoxides, acetonides, cyclic ketals and arylacetals.

TABLE A

wherein R^(1a) is C₁-C₆ alkyl.

“Alkyl” as used herein, unless stated to the contrary, is C₁-C₆hydrocarbon containing normal, secondary, tertiary or cyclic carbonatoms. Examples are methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃), 1-propyl(n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl, —CH(CH₃)₂),1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl,—CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃). Typical alkyls are methyl,ethyl, 1-propyl, and 2-propyl.

R² is H, F, —OR¹, or —OR⁶. Typically R² is H, or —OR¹, more typically, Hor —OH, still more typically, —OH.

R⁶ is

R³ is H or —CH₃. Typically R³ is —CH₃.

Each R⁴ of formula I and II is independently H or an amine protectinggroup, or both R⁴ groups of formula I are taken together to form acyclic amine protecting group. Typically, each R⁴ is H.

R⁴ amino protecting groups are described by Greene at pages 315-385.They include Carbamates (methyl and ethyl, 9-fluorenylmethyl,9(2-sulfo)fluoroenylmethyl, 9-(2,7-dibromo)fluorenylmethyl,2,7-di-t-buthyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl,4-methoxyphenacyl); Substituted Ethyl (2,2,2-trichoroethyl,2-trimethylsilylethyl, 2-phenylethyl, 1-(1-adamantyl)-1-methylethyl,1,1-dimethyl-2-haloethyl, 1,1-dimethyl-2,2-dibromoethyl,1,1-dimethyl-2,2,2-trichloroethyl, 1-methyl-1-(4-biphenylyl)ethyl,1-(3,5-di-t-butylphenyl)-1-methylethyl, 2-(2′- and 4′-pyridyl)ethyl,2-(N,N-dicyclohexylcarboxamido)ethyl, t-butyl, 1-adamantyl, vinyl,allyl, 1-isopropylallyl, cinnamyl, 4-nitrocinnamyl, 8-quinolyl,N-hydroxypiperidinyl, alkyldithio, benzyl, p-methoxybenzyl,p-nitrobenzyl, p-bromobenzyl, p-chorobenzyl, 2,4-dichlorobenzyl,4-methylsulfinylbenzyl, 9-anthrylmethyl, diphenylmethyl); Groups WithAssisted Cleavage (2-methylthioethyl, 2-methylsulfonylethyl,2-(p-toluenesulfonyl)ethyl, [2-(1,3-dithianyl)]methyl,4-methylthiophenyl, 2,4-dimethylthiophenyl, 2-phosphonioethyl,2-triphenylphosphonioisopropyl, 1,1-dimethyl-2-cyanoethyl,m-choro-p-acyloxybenzyl, p-(dihydroxyboryl)benzyl,5-benzisoxazolylmethyl, 2-(trifluoromethyl)-6-chromonylmethyl); GroupsCapable of Photolytic Cleavage (m-nitrophenyl, 3,5-dimethoxybenzyl,o-nitrobenzyl, 3,4-dimethoxy-6-nitrobenzyl,phenyl(o-nitrophenyl)methyl); Urea-Type Derivatives(phenothiazinyl-(10)-carbonyl, N′-p-toluenesulfonylaminocarbonyl,N′-phenylaminothiocarbonyl); Miscellaneous Carbamates (t-amyl, S-benzylthiocarbamate, p-cyanobenzyl, cyclobutyl, cyclohexyl, cyclopentyl,cyclopropylmethyl, p-decyloxybenzyl, diisopropylmethyl,2,2-dimethoxycarbonylvinyl, o-(N,N-dimethylcarboxamido)benzyl,1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl, 1,1-dimethylpropynyl,di(2-pyridyl)methyl, 2-furanylmethyl, 2-Iodoethyl, Isobornyl, Isobutyl,Isonicotinyl, p-(p′-Methoxyphenylazo)benzyl, 1-methylcyclobutyl,1-methylcyclohexyl, 1-methyl-1-cyclopropylmethyl,1-methyl-1-(3,5-dimethoxyphenyl)ethyl,1-methyl-1-(p-phenylazophenyl)ethyl, 1-methyl-1-phenylethyl,1-methyl-1-(4-pyridyl)ethyl, phenyl, p-(phenylazo)benzyl,2,4,6-tri-t-butylphenyl, 4-(trimethylammonium)benzyl,2,4,6-trimethylbenzyl); Amides (N-formyl, N-acetyl, N-choroacetyl,N-trichoroacetyl, N-trifluoroacetyl, N-phenylacetyl,N-3-phenylpropionyl, N-picolinoyl, N-3-pyridylcarboxamide,N-benzoylphenylalanyl, N-benzoyl, N-p-phenylbenzoyl); Amides WithAssisted Cleavage (N-o-nitrophenylacetyl, N-o-nitrophenoxyacetyl,N-acetoacetyl, (N′-dithiobenzyloxycarbonylamino)acetyl,N-3-(p-hydroxyphenyl)propionyl, N-3-(o-nitrophenyl)propionyl,N-2-methyl-2-(o-nitrophenoxy)propionyl,N-2-methyl-2-(o-phenylazophenoxy)propionyl, N-4-chlorobutyryl,N-3-methyl-3-nitrobutyryl, N-o-nitrocinnamoyl, N-acetylmethionine,N-o-nitrobenzoyl, N-o-(benzoyloxymethyl)benzoyl,4,5-diphenyl-3-oxazolin-2-one); Cyclic Imide Derivatives (N-phthalimide,N-dithiasuccinoyl, N-2,3-diphenylmaleoyl, N-2,5-dimethylpyrrolyl,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct, 5-substituted1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridonyl); N-Alkyl and N-Aryl Amines (N-methyl, N-allyl,N-[2-(trimethylsilyl)ethoxy]methyl, N-3-acetoxypropyl,N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl), Quaternary AmmoniumSalts, N-benzyl, N-di(4-methoxyphenyl)methyl, N-5-dibenzosuberyl,N-triphenylmethyl, N-(4-methoxyphenyl)diphenylmethyl,N-9-phenylfluorenyl, N-2,7-dichloro-9-fluorenylmethylene,N-ferrocenylmethyl, N-2-picolylamine N′-oxide), Imine Derivatives(N-1,1-dimethylthiomethylene, N-benzylidene, N-p-methoxybenylidene,N-diphenylmethylene, N-[(2-pyridyl)mesityl]methylene,N,(N′,N′-dimethylaminomethylene, N,N′-isopropylidene,N-p-nitrobenzylidene, N-salicylidene, N-5-chlorosalicylidene,N-(5-chloro-2-hydroxyphenyl)phenylmethylene, N-cyclohexylidene); EnamineDerivatives (N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)); N-Metal Derivatives(N-borane derivatives, N-diphenylborinic acid derivatives,N-[phenyl(pentacarbonylchromium- or -tungsten)]carbenyl, N-copper orN-zinc chelate); N-N Derivatives (N-nitro, N-nitroso, N-oxide); N-PDerivatives (N-diphenylphosphinyl, N-dimethylthiophosphinyl,N-diphenylthiophosphinyl, N-dialkyl phosphoryl, N-dibenzyl phosphoryl,N-diphenyl phosphoryl); N-Si Derivatives; N-S Derivatives; N-SulfenylDerivatives (N-benzenesulfenyl, N-o-nitrobenzenesulfenyl,N-2,4-dinitrobenzenesulfenyl, N-pentachlorobenzenesulfenyl,N-2-nitro-4-methoxybenzenesulfenyl, N-triphenylmethylsulfenyl,N-3-nitropyridinesulfenyl); and N-sulfonyl Derivatives(N-p-toluenesulfonyl, N-benzenesulfonyl,N-2,3,6-trimethyl-4-methoxybenzenesulfonyl,N-2,4,6-trimethoxybenzenesulfonyl,N-2,6-dimethyl-4-methoxybenzenesulfonyl, N-pentamethylbenzenesulfonyl,N-2,3,5,6,-tetramethyl-4-methoxybenzenesulfonyl,N-4-methoxybenzenesulfonyl, N-2,4,6-trimethylbenzenesulfonyl,N-2,6-dimethoxy-4-methylbenzenesulfonyl,N-2,2,5,7,8-pentamethylchroman-6-sulfonyl, N-methanesulfonyl,N-β-trimethylsilyethanesulfonyl, N-9-anthracenesulfonyl,N-4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonyl, N-benzylsulfonyl,N-trifluoromethylsulfonyl, N-phenacylsulfonyl).

R⁵ is H, —CH₃ or —C≡C—CH₃. Typically R⁵ is —CH₃.

In another embodiment of the invention, when the compound is of: formulaI wherein each of R¹, R², R³, R⁴, and R⁵ are H; or formula II whereineach of R¹, R², R³, and R⁴ are H, and R⁵ is —CH₃; then the compound isnot of the formula:

In another embodiment the compound is of formula III or IV.

In another embodiment the compound is of formula III or IV, with theproviso that the compound is not of formula I wherein each of R¹, R²,R³, R⁴, and R⁵ are H; or of formula II wherein each of R¹, R², R³, andR⁴ are H, and R⁵ is —CH₃.

In another embodiment the compound is of formula I or II wherein R³ is—CH₃.

In another embodiment the compound is of formula I.

In another embodiment the compound is of formula III.

In another embodiment the compound is of formula III with the provisothat the compound is not of formula III wherein each of R¹, R², R³, R⁴,and R⁵ are H. Typically, R³ is —CH₃.

In another embodiment the compound is of the formula:

In another embodiment the composition of formula V wherein:

R² is H and R⁵ is H;

R² is H and R⁵ is —CH₃;

R² is H and R⁵ is —C≡C—CH₃;

R² is —OH and R⁵ is H;

R² is —OH and R⁵ is —CH₃; or

R² is —OH and R⁵ is —C≡C—CH₃. Typically R² is H and R⁵ is —CH₃.

Stereoisomers

The compounds of the invention are enriched or resolved optical isomersat any or all asymmetric atoms. For example, the chiral centers apparentfrom the depictions are provided as the chiral isomers or racemicmixtures. Both racemic and diasteromeric mixtures, as well as theindividual optical isomers isolated or synthesized, substantially freeof their enantiomeric or diastereomeric partners, are all within thescope of the invention. The racemic mixtures are separated into theirindividual, substantially optically pure isomers through well-knowntechniques such as, for example, the separation of diastereomeric saltsformed with optically active adjuncts, e.g., acids or bases followed byconversion back to the optically active substances. By way of exampleand not limitation, methods of resolving individual enantiomers aredescribed in “Enantiomers, Racemates, and resolutions”, Jean Jacques,Andre Collet, and Samuel H. Wilen (Krieger Publishing Company, Malabar,Fla., 1991, ISBN 0-89464-618-4). In particular, Part 2, Resolution ofEnantiomer Mixture, pages 217-435; more particularly, section 4,Resolution by Direct Crystallization, pages 217-251, section 5,Formation and Separation of Diastereomers, pages 251-369, section 6,Crystallization-Induced Asymmetric Transformations, pages 369-378, andsection 7, Experimental Aspects and Art of Resolutions, pages 378-435;still more particularly, section 5.1.4, Resolution of Alcohols.Transformation of Alcohols into Salt-Forming Derivatives, pages 263-266,section 5.2.3 Covalent Derivatives of Alcohols, Thiols, and Phenols,pages 332-335, and section 5.2.7, Chromatographic Behavior of CovalentDiastereomers, pages 348-354, are cited as examples of the skill of theart. In most instances, the desired optical isomer is synthesized bymeans of stereospecific reactions, beginning with the appropriatestereoisomer of the desired starting material.

Exemplary Enumerated Compounds

By way of example and not limitation, embodiment compounds are namedbelow in tabular format (Table 5). Generally, each compound is depictedas a substituted nucleus in which the nucleus is designated by capitalletter and each substituent is designated in order by number or capitalletter. Table 1 is a schedule of nuclei. Each nucleus is given analphabetical designation from Table 1, and this designation appearsfirst in each compound name. Similarly, Tables 2, 3, and 4 list theselected R², R³ and R⁵ substituents, again by number or capital letterdesignation. Accordingly, each named compound will be depicted by acapital letter designating the nucleus from Table 1, followed by anumber designating the R² substituent, a capital letter designating theR³ substituent, and a number designating the R⁵ substituent. Thus, thecompound is represented by A.1.B.2.

TABLE 1

A

B

TABLE 2

1

2

3

4

5

6

7

8

TABLE 3

A

B

TABLE 4

1

2

3

TABLE 5 A.1.A.1; A.1.A.2; A.1.A.3; A.1.B.1; A.1.B.2; A.1.B.3; A.2.A.1;A.2.A.2; A.2.A.3; A.2.B.1; A.2.B.2; A.2.B.3; A.3.A.1; A.3.A.2; A.3.A.3;A.3.B.1; A.3.B.2; A.3.B.3; A.4.A.1; A.4.A:2; A.4.A.3; A.4.B.1; A.4.B.2;A.4.B.3; A.5.A.1; A.5.A.2; A.5.A.3; A.5.B.1; A.5.B.2; A.5.B.3; A.6.A.1;A.6.A.2; A.6.A.3; A.6.B.1; A.6.B.2; A.6.B.3; A.7.A.1; A.7.A.2; A.7.A.3;A.7.B.1; A.7.B.2; A.7.B.3; A.8.A.1; A.8.A.2; A.8.A.3; A.8.B.1; A.8.B.2;A.8.B.3; B.1.A.1; B.1.A.2; B.1.A.3; B.1.B.1; B.1.B.2; B.1.B.3; B.2.A.1;B.2.A.2; B.2.A.3; B.2.B.1; B.2.B.2; B.2.B.3; B.3.A.1; B.3.A.2; B.3.A.3;B.3.B.1; B.3.B.2; B.3.B.3; B.4.A.1; B.4.A.2; B.4.A.3; B.4.B.1; B.4.B.2;B.4.B.3; B.5.A.1; B.5.A.2; B.5.A.3; B.5.B.1; B.5.B.2; B.5.B.3; B.6.A.1;B.6.A.2; B.6.A.3; B.6.B.1; B.6.B.2; B.6.B.3; B.7.A.1; B.7.A.2; B.7.A.3;B.7.B.1; B.7.B.2; B.7.B.3; B.8.A.1; B.8.A.2; B.8.A.3; B.8.B.1; B.8.B.2;B.8.B.3

Salts and Hydrates

The compositions of this invention optionally comprise salts of thecompounds herein, for example, Na⁺, Li⁺, K⁺, Ca⁺⁺ and Mg⁺⁺. Such saltsmay include those derived by combination of appropriate cations such asalkali and alkaline earth metal ions or ammonium and quaternary aminoions with an acid anion moiety. Monovalent salts are preferred if awater soluble salt is desired.

Metal salts typically are prepared by reacting the metal hydroxide witha compound of this invention. Examples of metal salts which are preparedin this way are salts containing Li⁺, Na⁺, and K⁺. A less soluble metalsalt can be precipitated from the solution of a more soluble salt byaddition of the suitable metal compound.

In addition, salts may be formed from acid addition of certain organicand inorganic acids, e.g., HCl, HBr, H₂SO₄, or organic sulfonic acids,to basic centers, typically amines. Finally, it is to be understood thatthe compositions herein comprise compounds of the invention in theirun-ionized, as well as zwitterionic form, and combinations withstoiochimetric amounts of water as in hydrates.

Also included within the scope of this invention are the salts of theparental compounds with one or more amino acids. Any of the amino acidsdescribed above are suitable, especially the naturally-occurring aminoacids found as protein components, although the amino acid typically isone bearing a side chain with a basic or acidic group, e.g., lysine,arginine or glutamic acid, or a neutral group such as glycine, serine,threonine, alanine, isoleucine, or leucine.

Oligonucleotides

Another embodiment of the present invention is directed to oligomerscapable of triple helix formation comprising a multiplicity ofnucleosides linked by internucleoside linkages wherein at least onenucleoside is a 2-aminopyridine and 2-pyridone C-nucleosides.

The principals of triplex binding and assays utilizing triplex bindinghave been described in detail elsewhere and will not be repeated here.By way of example and not limitation, see Froehler, B. and Jones, R. J.,U.S. Pat. No. 5,484,908, Jan. 16, 1996, in particular column 1, line 1,to column 3, line 55, and Cantor, C. R. and Smith, C. L., U.S. Pat. No.5,482,836, Jan. 9, 1996, in particular column 1, line 30, to column 2,line 58.

An oligonucleotide capable of binding specifically to a duplexoligonucleotide is an oligonucleotide that binds in a triplex mode to agiven target duplex. Specificity is determined by the particularapplication. Generally, selectivity is expressed as the ratio ofoligonucleotide bound to the target section of duplex vs.oligonucleotide bound to another section of duplex. Typically >1:1binding is selective binding, more typically >10:1, still moretypically >100:1. In some applications binding ratios higher than 1000:1or 10000:1 are obtained.

As used herein, oligonucleotide means single stranded or double strandedDNA or RNA, and analogs of DNA or RNA and plasmids comprisingoligonucleotides. In general, relatively large nucleic acids such asplasmids or mRNAs will carry one or more genes that are to be expressedin a transfected cell, while comparatively small nucleic acids, i.e.,typical oligonucleotides, will comprise (1) a base sequence that iscomplementary (via Watson Crick or Hoogsteen binding) to a DNA or RNAsequence present in the cell or (2) a base sequence that permitsoligonucleotide binding to a molecule inside a cell such as a peptide,protein or glycoprotein. Exemplary RNAs include ribozymes and antisenseRNA sequences that are complementary to a target RNA sequence in a cell.

Oligonucleotides include single stranded unmodified DNA or RNAcomprising (a) the purine or pyrimidine bases guanine, adenine,cytosine, thymine and/or uracil; (b) ribose or deoxyribose; and (c) aphosphodiester group that linkage adjacent nucleoside moieties.Oligonucleotides typically comprise 2 to about 100 or 3 to about 100linked nucleosides. Typical oligonucleotides comprise size ranges suchas 2-10, 2-15, 2-20, 2-25, 2-30, 7-15, 7-20, 7-30 or 7-50 linkednucleotides. Oligonucleotides can be linear, circular, branched ordouble-stranded. Oligonucleotides are usually linear with uniformpolarity but, when regions of inverted polarity are present, suchregions comprise no more than one polarity inversion per 10 nucleotides.One inversion per 20 nucleotides is typical. Antisense oligonucleotidesgenerally will comprise a sequence of about 7-50 bases, usually about8-30 bases. The oligonucleotide base sequence is usually complementaryor substantially complementary to a cognate DNA or RNA base sequencepresent in the cell. The size of nucleic acid of the invention islimited only by the size of molecules that reasonably can be prepared byconventional means.

Oligonucleotides also include DNA or RNA comprising one or more covalentmodifications. Covalent modifications include (a) replacement of thephosphodiester group with a nonphosphorus moiety such as —O—CH₂—O—,—S—CH₂—O— or —O—CH₂—S—, and (c) replacement of the phosphodiester groupwith a phosphate analog such as —O—P(S)(O)—O— (phosphorothioatelinkage), —O—P(S)(S)—O—, —O—P(CH₃)(O)—O— or —O—P(NHR¹³)(O)—O— where R¹³is alkyl (C₁₋₆), or an alkyl ether (C₁₋₆). Oligonucleotides includemodified oligonucleotides having a substitution at about 20-100%, moreoften about 40-100% and usually about 80%-100% of the phosphodiestergroups in unmodified DNA or RNA. Such modified oligonucleotidesoptionally also have 20-100%, more often about 40-100% or about 80%-100%of the pyrimidine bases substituted with 5-(1-propynyl)uracil or5-(1-propynyl)cytosine. Oligonucleotides include covalent modificationor isomers of ribose or deoxyribose such as morpholino, arabinose,2′-fluororibose, 2′-fluoroarabinose, 2′-O-methylribose or2′-O-allylribose. Oligonucleotides and methods to synthesize them havebeen described (for example see: PCT/US90/03138, PCT/US90/06128,PCT/US90/06090, PCT/US90/06110, PCT/US92/03385, PCT/US91/08811, PCT/US91/03680, PCT/US91 /06855, PCT/US91 /01141, PCT/US92/10115,PCT/US92/10793, PCT/US93/05110, PCT/US93/05202, PCT/US92/04294,US94/04013, W086/05518, W089/12060, W091/08213, W090/15065, W091/15500,W092/02258, W092/20702, W092/20822, W092/20823, U.S. Pat. No. 5,214,136and Uhlmann et al. Chem. Rev. (1990) 90:543.

Linkage means a moiety suitable for coupling adjacent nucleomonomers andincludes both phosphorus-containing moieties and nonphosphorus-containing moieties such as formacetal, thioformacetal,riboacetal and the like. A linkage usually comprises 2 or 3 atomsbetween the 5′ position of a nucleotide and the 2′ or 3′ position of anadjacent nucleotide. Linkages between the 5′ and 2′ positions willusually not contain phosphorus.

A purine or pyrimidine base means a heterocyclic moiety suitable forincorporation into an oligonucleotide. It can be in the α or β anomerconfiguration. Purine or pyrimidine bases are moieties that bind tocomplementary nucleic acid sequences by Watson-Crick or Hoogsteen basepair rules. Bases need not always increase the binding affinity of anoligonucleotide for binding to its complementary sequence at least ascompared to bases found in native DNA or RNA. However, such modifiedbases preferably are not incorporated into an oligomer to such an extentthat the oligonucleotide is unable to bind to complementary sequences toproduce a detectably stable duplex or triplex. Purine or pyrimidinebases usually pair with a complementary purine or pyrimidine base via 1,2 or 3 hydrogen bonds. Such purine or pyrimidine bases are generally thepurine, pyrimidine or related heterocycles shown in formulas G-I.

wherein R³⁵ is H, —OH, F, Cl, Br, I, —OR³⁶, —SH, —SR³⁶, —NH₂, or —NHR³⁷;

R³⁶ is C₁-C₆ alkyl (including —CH₃, —CH₂CH₃ and —C₃H₇), —CH₂CCH(2-propynyl) and —CH₂CHCH₂;

R³⁷ is C₁-C₆ alkyl including —CH₃, —CH₂CH₃, —CH₂CCH, —CH₂CHCH₂, —C₃H₇;

R³⁸ is N, CF, CCl, CBr, CI, CR³⁹ or CSR³⁹, COR³⁹;

R³⁹ is H, C₁-C₉ alkyl, C₂-C₉ alkenyl, C₂-C₉ alkynyl or C₇-C₉ aryl-alkylunsubstituted or substituted by OH, O, N, F, Cl, Br or I including —CH₃,—CH₂CH₃, —CHCH₂, —CHCHBr, —CH₂CH₂Cl, —CH₂CH₂F, —CH₂CCH, —CH₂CHCH₂,—C₃H₇, —CH₂OH, —CH₂OCH₃, C—H₂OC₂H₅, —CH₂OCCH, —CH₂OCH₂CHCH_(2, —CH)₂C₃H₇, —CH₂CH₂OH, —CH₂CH₂OCH₃, —CH₂CH₂OC₂H₅, —CH₂CH₂OCCH,—CH₂CH₂OCH₂CHCH₂, —CH₂CH₂OC₃H₇;

R⁴⁰ is N, CBr, CI, CCl, CH, C(CH₃), C(CH₂CH₃) or C(CH₂CH₂CH₃);

R⁴¹ is N, CH, CBr, CCH₃, CCN, CCF₃, CC≡CH or CC(O)NH₂;

R⁴² is H, OH, NH₂, SH, SCH₃, SCH₂CH₃, SCH₂CCH, SCH₂CHCH₂, SC₃H₇,NH(CH₃), N(CH₃)₂, NH(CH₂CH₃), N(CH₂CH₃)₂, NH(CH₂CCH), NH(CH₂CHCH₂),NH(C₃H₇) or F, Cl, Br or I;

R⁴³ is H, OH, F, Cl, Br, I, SCH₃, SCH₂CH₃, SCH₂CCH, SCH₂CHCH₂, SC₃H₇,OR¹⁶, NH₂, or NHR³⁷; and

R⁴⁴ is O, S or Se.

Exemplary bases include adenine, cytosine, guanine, hypoxanthine,inosine, thymine, uracil, xanthine, 2-aminopurine, 2,6-diaminopurine,5-(4-methylthiazol-2-yl)uracil, 5-(5-methylthiazol-2-yl)uracil,5-(4-methylthiazol-2-yl)cytosine, 5-(5-methylthiazol-2-yl)cytosine andthe like.

Also included are alkylated or alkynylated bases having substitutionsat, for example, the 5 position of pyrimidines that results in apyrimidine base other than uracil, thymine or cytosine, i.e.,5-methylcytosine, 5-(1-propynyl)cytosine, 5-(1-butynyl)cytosine,5-(1-butynyl)uracil, 5-(1-propynyl)uracil and7-(1-propynyl)-7-deazaguanine. Base analogs and their use in oligomershave been described (see for example, U.S. application Ser. No.08/123,505; WO 92/10115; WO 91/08811; WO 92/09195; WO 93/10820; WO92/09705; WO 92/02258; Nikiforov, T. T., et al, Tet Lett (1992)33:2379-2382; Clivio, P., et al, Tet Lett (1992) 33:65-68; Nikiforov, T.T., et al, Tet Lett (1991) 32:2505-2508; Xu, Y.-Z., et al, Tet Lett(1991) 32:2817-2820; Clivio, P., et al, Tet Lett (1992) 33:69-72;Connolly, B. A., et al, Nucl Acids Res (1989) 17:4957-4974).Oligonucleotides having varying amounts of bases analogs such as5-methylcytosine, 5-(1-propynyl)cytosine, 5-(1-butynyl)cytosine or5-(1-butynyl)uracil, 5-(1-propynyl)uracil or7-(1-propynyl)-7-deazaguanine, e.g., about 20-80%, usually about 80-100%of the natural bases are substituted with the corresponding analogs.

Triplex-Affinity Capture

Another aspect of the invention is directed to a method for purifyingintact DNA using intermolecular triple-helix formation and solid phaseseparation. The details of the method have been described elsewhere andwill not be repeated here. By way of example and not limitation, seeCantor, C. R. and Smith, C. L., U.S. Pat. No. 5,482,836, Jan. 9, 1996.In this triplex-affinity capture (TAC) method, the DNA being detected inthe assay is intact double stranded DNA and the method can be used tocapture sequence specific plasmid DNAs. Essentially, the target DNAsequence is a double stranded homopurine-homopyrimidine helix.Nevertheless, the method may be extended by the use of somepermissiveness mismatches in triple-helix formation (Griffin, L. C., etal. Science (1989) 245:967-971 and Belotserkovskii, B. D., et al.Nucleic Acids Res. (1990) 18:6621-6624), alternate strand triple-helixformation (Horne, D. A., et al. J. M. Chem. Soc. (1990) 112:2435-2438),other types of triple-helices (Cooney, M., et al. Science (1988)241:456459; Kohwi, Y., et al. Proc. Natl. Acad. Sci. USA (1988)85:3781-3785; Letai, A. G., et al. Biochemistry (1988) 27:9108-9112;Bernues, J., et al. EMBO J. (1989) 8:2087-2094; Beal, P. A., et al.Science (1991) 251:1360-1363; Pilch, D. S., et al. Biochemistry (1991)30:6081-6087; Orson, F. M., et al. Nucleic Acids Res. (1991)19:3435-3441), including ones formed by recombinase proteins (Hsieh, P.,et al. Genes Dev. (1990) 4:1951-1963; Rao, B. J., et al. Proc. Natl.Acad. Sci. USA (1991) 88:2984-2988) and artificial base analogs.

The TAC procedure of the invention is especially appropriate forisolating (dT-dC)_(n)·(dG-dA)_(n) dinucleotide repeats from humangenome. This sequence is a member of so-called “microsatellite” DNAsdistributed throughout mammalian genomes (Manor, H., et al. J. Mol.Evol. (1988) 27:96-101; Wong, A. K. C., et al. Chromosoma (1990)99:344-351). It is often hyper-variable in the number of repeat units(n) from individual-to-individual and thus provides highly informativeDNA markers for genetic linkage mapping (Tautz, D., Nucleic Acids Res.(1989) 17:6463-6471; Love, J. M., et al. Nucleic Acids Res. (1990)18:4123-4130; Moore, S. S., et al. Genomics (1991) 10:654-660; Weber, J.L. (1990) in Genome Analysis, eds. Davies, K. E. et al. (Cold SpringHarbor Lab., Cold Spring Harbor, N.Y.). Vol. 1, pp. 159-181). The TACmethod may also be used for the effective enrichment of triplex formingsingle copy sequences from yeast and more complex genomes using theappropriate probes. The use of G and a novel artificial base analog(Kiessling, L. L., et al. Biochemistry (1992) 31:2829-2834) in the thirdstrand has broadened the triplex recognition capability to allow one tofind a target for TAC in natural non-tagged sequences with much ease.

The TAC method for purifying a particular double strand of DNA comprisescontacting the sample with an oligonucleotide of the invention coupledeither directly or indirectly to a first recognition molecule of aspecific molecular recognition system. The oligonucleotide is designedto specifically form a triple helix with the target DNA. Methods fordesigning such oligonucleotides depend on the target DNA. Acceptablemethods are set forth in Kiessling, L. L., et al. Biochemistry (1992)31:2829-2834; Durland, R. H., et al. Biochemistry (1991) 30:9246-9255;Beal, P. A., et al. Nucleic Acids Res. (1992) 20:2773-2776;Giovannangeli, C., et al. Proc. Natl. Acad. Sci. (1992) 89:8631-8635;Beal, P. A., et al., J. Am. Chem. Soc. (1992) 114:4976-4982.Oligonucleotides which contain deoxyuracil for thymine at least alongpart of the chain are acceptable oligonucleotides. Oligonucleotidebackbone analogs such as polyamide nucleic acids and phosphotriesterswill form a triplex with double stranded DNA and can also be used in theTAC method of the invention.

The triplexes formed between the specific oligonucleotides of theinvention and the target DNA molecules containing the correspondinghomopurine-homopyrimidine sequences are subsequently contacted with asolid carrier to which is either directly or indirectly fixed a secondrecognition molecule belonging to the same molecular recognition systemas the first recognition molecule coupled to the oligonucleotide. Thesecond recognition molecule is a molecule which will specifically bindto the first recognition molecule. The solid phase is subsequentlyseparated from the reaction medium where the binding occurred andtherefore is also separated from any remaining non-triplexed nucleicacids. Finally, the target DNAs are recovered in intact double strandedform by treating the separated solid phase bearing the triple-helix witha reagent that breaks the bonds between the oligonucleotide and theparticular DNA but not between the double helix DNA. The particular DNAis then recovered.

Using several methods well-known in the art including electrophoresisand fluorometry, the TAC method can also be used to determine thepresence or absence of a particular double stranded DNA in a sample bytesting for the presence of the particular DNA in the eluate after thetriple helix separation step.

Another aspect of the invention is directed to improvments in atriplex-affinity capture purification wherein the improvement comprisesemploying as the coupled oligonucleotide an oligonucleotide containingone or more of the modified oligonucleosides (i.e. a 2-aminopyridine and2-aminopyridone C-nucleoside) of the present invention. One example ofthe triplex-affinity capture purification of this embodiment isdescribed in Cantor, C. R. and Smith, C. L., U.S. Pat. No. 5,482,836,Jan. 9, 1996, in particular one or more of the methods described atcolumn 21, line 22, to column 26, line 39.

Assays

Another embodiment of the present invention is directed to methods ofdetecting the presence, absence or amount of a particular DNA duplex ina sample suspected of containing DNA comprising contacting the samplewith an oligomer of the invention under conditions wherein a triplehelix is formed between the oligomer and the particular DNA duplex.

The conventional aspects of oligonucleotide hybridization assays, inparticular, the principals of triplex binding and assays utilizingtriplex binding, have been described in detail elsewhere and will not berepeated here. By way of example and not limitation, see Froehler, B.and Jones, R. J., U.S. Pat. No. 5,484,908, Jan. 16, 1996, in particularcolumn 1, line 1, to column 3, line 55, and column 20, line 38, tocolumn 21, line 18.

Generally, the oligomers of the invention may be used as diagnosticreagents to detect the presence or absence of the target gene sequencesto which they specifically bind. Such diagnostic tests are conducted byhybridization through either double or triple helix formation which isthen detected by conventional means. For example, the oligomers may belabeled using radioactive, fluorescent, or chromogenic labels and thepresence of label bound to solid support detected. Alternatively, thepresence of a double or triple helix may be detected by antibodies whichspecifically recognize these forms. Means for conducting assays usingsuch oligomers as probes are generally known.

The use of oligomers containing the modified bases as diagnostic agentsby triple helix formation is advantageous since triple helices formunder mild conditions and the assays may thus be carried out withoutsubjecting test specimens to harsh conditions. Diagnostic assays basedon detection of RNA for identification of bacteria, fungi or protozoasequences often require isolation of RNA from samples or organisms grownin the laboratory, which is laborious and time consuming; as RNA isextremely sensitive to ubiquitous nucleases.

The oligomer probes may also incorporate additional modifications suchas altered internucleotide linkages that render the oligomer especiallynuclease stable, and would thus be useful for assays conducted in thepresence of cell or tissue extracts which normally contain nucleaseactivity. Oligonucleotides containing terminal modifications oftenretain their capacity to bind to complementary sequences without loss ofspecificity (Uhlmann et al., Chemical Reviews (1990) 90:543-584). As setforth above, the invention probes may also contain linkers that permitspecific binding to alternate DNA strands by incorporating a linker thatpermits such binding (Horne et al., J Am Chem Soc (1990) 112:2435-2437).

Incorporation of base analogs of the present invention into probes thatalso contain covalent crosslinking agents has the potential to increasesensitivity and reduce background in diagnostic or detection assays. Inaddition, the use of crosslinking agents will permit novel assaymodifications such as (1) the use of the crosslink to increase probediscrimination, (2) incorporation of a denaturing wash step to reducebackground and (3) carrying out hybridization and crosslinking at ornear the melting temperature of the hybrid to reduce secondary structurein the target and to increase probe specificity. Modifications ofhybridization conditions have been previously described (Gamper et al.,Nucleic Acids Res (1986) 14:9943).

The conventional aspects of oligonucleotide hybridization assays arewell known and will not be repeated here.

Additional Uses for the Compounds of This Invention

The compounds of the invention are polyfunctional. As such theyrepresent a unique class of monomers for the synthesis of polymers. Byway of example and not limitation, the polymers prepared from thecompounds of this invention include polyamides, polyesters and mixedpolyester-polyamides.

The present compounds are used as monomers to provide access to polymershaving unique pendent functionalities. The compounds of this inventionare useful as comonomers with monomers which do not fall within thescope of the invention. Polymers of the compounds of this invention willhave utility as cation exchange agents (polyesters or polyamides) in thepreparation of molecular sieves (polyamides), textiles, fibers, films,formed articles and the like. Polymers are prepared by any conventionalmethod, for example, by cross-linking an —OH or —NH₂ group of thecompounds of the invention with a diacid comonomer. The preparation ofthese polymers from the compounds of the invention is conventional perse.

The compounds of the invention are also useful as a unique class ofpolyfunctional surfactants. Particularly when R¹ or R² do not containhydrophilic substituents and are, for example, alkyl or alkoxy, thecompounds have the properties of bi-functional surfactants. As such theyhave useful surfactant, surface coating, emulsion modifying, rheologymodifying and surface wetting properties.

As polyfunctional compounds with defined geometry and carryingsimultaneously polar and non-polar moieties, the compounds of theinvention are useful as a unique class of phase transfer agents. By wayof example and not limitation, the compounds of the invention are usefulin phase transfer catalysis and liquid/liquid ion extraction (LIX).

The compounds of the invention optionally contain asymmetric carbonatoms. As such, they are a unique class of chiral auxiliaries for use inthe synthesis or resolution of other optically active materials. Forexample, a racemic mixture of carboxylic acids can be resolved into itscomponent enantiomers by: 1) forming a mixture of diastereomeric estersor amides with a compound of the invention containing an —OH or —NH₂group; 2) separating the diastereomers; and 3) hydrolyzing the esterstructure. Further, such a method can be used to resolve the compoundsof the invention themselves if optically active acids are used insteadof racemic starting materials.

The compounds of this invention are useful as linkers or spacers inpreparing affinity absorption matrices, immobilized enzymes for processcontrol, or immunoassay reagents. The compounds herein contain amultiplicity of functional groups that are suitable as sites forcross-linking desired substances. For example, it is conventional tolink affinity reagents such as hormones, peptides, antibodies, drugs,and the like to insoluble substrates. These insolublized reagents areemployed in known fashion to absorb binding partners for the affinityreagents from manufactured preparations, diagnostic samples and otherimpure mixtures. Similarly, immobilized enzymes are used to performcatalytic conversions with facile recovery of enzyme. Bifunctionalcompounds are commonly used to link analytes to detectable groups inpreparing diagnostic reagents.

Many functional groups in the compounds of this invention are suitablefor use in cross-linking. For example, —OH and —NH₂ groups. Suitableprotection of reactive groups will be used where necessary whileassembling the cross-linked reagent to prevent polymerization of thebifunctional compound of this invention. In general, the compounds hereare used by linking them through hydroxyl or amino groups to carboxylicor phosphonic acid groups of the first linked partner, then covalentlybonding to the other binding partner through another —OH or —NH₂ group.For example a first binding partner such as a steroid hormone is reactedto form an amide bond with the —NH₂ group of a compound of thisinvention and then this conjugate is cross-linked through a hydroxyl tocyanogen bromide activated Sepaharose, whereby immobilized steroid isobtained. Other chemistries for conjugation are well known. See forexample Maggio, Enzyme Immunoassay (CRC, 1988, pp 71-135) and referencescited therein.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake the compounds and compositions of the invention and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to insure accuracy with respect tonumbers used (e.g., amounts, temperatures, etc.), but some experimentalerrors and deviations should be taken into account. Unless indicatedotherwise, parts are parts by weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Synthesis of 2-Aminopyridine and 2-pyridone C-nucleosides

The invention is also directed to methods of making the compositions ofthe invention. The compositions are prepared by any of the applicabletechniques of organic synthesis. Many such techniques are well known inthe art. However, many of the known techniques are elaborated in“Compendium of Organic Synthetic Methods” (John Wiley & Sons, New York),Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T.Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and LeroyWade, 1977; Vol. 4, Leroy G. Wade, jr., 1980; Vol. 5, Leroy G. Wade,Jr., 1984; and Vol. 6, Michael B. Smith; as well as March, J., “AdvancedOrganic Chemistry, Third Edition”, (John Wiley & Sons, New York, 1985),“Comprehensive Organic Synthesis. Selectivity, Strategy & Efficiency inModern Organic Chemistry. In 9 Volumes”, Barry M. Trost, Editor-in-Chief(Pergamon Press, New York, 1993 printing).

A number of exemplary methods for the preparation of the compositions ofthe invention are provided below. These methods are intended toillustrate the nature of such preparations are not intended to limit thescope of applicable methods.

Generally, the reaction conditions such as temperature, reaction time,solvents, workup procedures, and the like, will be those common in theart for the particular reaction to be performed. The cited referencematerial, together with material cited therein, contains detaileddescriptions of such conditions. Typically the temperatures will be−100° C. to 200° C., solvents will be aprotic or protic, and reactiontimes will be 10 seconds to 10 days. Workup typically consists ofquenching any unreacted reagents followed by partition between awater/organic layer system (extraction) and separating the layercontaining the product.

Oxidation and reduction reactions are typically carried out attemperatures near room temperature (about 20° C.), although for metalhydride reductions frequently the temperature is reduced to 0° C. to−100° C., solvents are typically aprotic for reductions and may beeither protic or aprotic for oxidations. Reaction times are adjusted toachieve desired conversions.

Condensation reactions are typically carried out at temperatures nearroom temperature, although for non-equilibrating, kinetically controlledcondensations reduced temperatures (0° C. to −100° C.) are also common.Solvents can be either protic (common in equilibrating reactions) oraprotic (common in kinetically controlled reactions).

Standard synthetic techniques such as azeotropic removal of reactionby-products and use of anhydrous reaction conditions (e.g. inert gasenvironments) are common in the art and will be applied when applicable.

One exemplary method of making the nucleosides of the invention isdepicted in Scheme 1. R¹, R², R³, R⁴ and R⁵ are as described above. TheR¹ group on the 5′ O is typically H and the R¹ group on the 3′ O isusually a hydroxy protecting group stable to the conditions used toprepare compound 3. Typically the protecting group is an acid labile,base stable protecting group. Each R⁴ is usually H but may be an amineprotecting group. R² is usually H.

Iodopyridine 1 is reacted with olefin 2 to produce compound 3. The 10reaction is typically accomplished using a metal catalyst, moretypically a Palladium catalyst. A suitable solvent is optionallypresent. Typically an amine solvent (C₁-C₆ trialkyl amine is usual),optionally in the presence of a cosolvent such as CH₃CN. For example thereaction can be performed using Pd(OAc)₂/Ph₃As in Bu₃N/CH₃CN.

Compound 3 is reacted to form compound 4. Typically the protecting groupis removed. For example if the protecting group is a silyl ether, acidoptionally in the presence of a fluoride source is employed.HOAc/TBAF/THF is typical for a TBDPSi group.

The reduction of compound 4 to prepare compound 5 is carried out by anyof the usual methods. For example a hydroborating agent such asNaBH(OAc)₃ is typical.

Another embodiment of the invention is the method of Scheme 1 whereinthe 2-aminopyridine compound 1 is replaced with the corresponding2-pyridone to produce the 2-pyridone version of compound 5.

Modifications of each of the above schemes leads to various analogs ofthe specific exemplary materials produced above. The above citedcitations describing suitable methods of organic synthesis areapplicable to such modifications.

In each of the above exemplary schemes it may be advantageous toseparate reaction products from one another and/or from startingmaterials. The desired products of each step or series of steps isseparated and/or purified (hereinafter separated) to the desired degreeof homogeneity by the techniques common in the art. Typically suchseparations involve multiphase extraction, crystallization from asolvent or solvent mixture, distillation, sublimation, orchromatography. Chromatography can involve any number of methodsincluding, for example, size exclusion or ion exchange chromatography,high, medium, or low pressure liquid chromatography, small scale andpreparative thin or thick layer chromatography, as well as techniques ofsmall scale thin layer and flash chromatography.

Another class of separation methods involves treatment of a mixture witha reagent selected to bind to or render otherwise separable a desiredproduct, unreacted starting material, reaction by product, or the like.Such reagents include adsorbents or absorbents such as activated carbon,molecular sieves, ion exchange media, or the like. Alternatively, thereagents can be acids in the case of a basic material, bases in the caseof an acidic material, binding reagents such as antibodies, bindingproteins, selective chelators such as crown ethers, liquid/liquid ionextraction reagents (LIX), or the like.

Selection of appropriate methods of separation depends on the nature ofthe materials involved. For example, boiling point, and molecular weightin distillation and sublimation, presence or absence of polar functionalgroups in chromatography, stability of materials in acidic and basicmedia in multiphase extraction, and the like. One skilled in the artwill apply techniques most likely to achieve the desired separation.

EXAMPLES Example 14-(2′-Deoxy-β-D-ribofuranosyl)-2-amino-3,6-dimethylpyridine

A mixture of palladium (II) acetate (0.029 g, 0.13 mmol),triphenylarsine (0.079 g, 0.26 mmol) and CH₃CN (8 mL) was stirred underan Argon atmosphere at room temperature. A solution of1,4-anhydro-2-deoxy-3-O-[(1,1-dimethylethyl)diphenylsilyl]-D-erythro-pent-1-en-itol(0.513 g, 1.45 mmol) and tributylamine (0.405 mL, 1.7 mmol) in CH₃CN (8mL) was added followed immediately by addition of2-amino-3,6-dimethyl-4-iodopyridine (0.322 g, 1.3 mmol). The mixture wasstirred at 60° C. for 15 h and then cooled to 0° C. Added was aceticacid (0.245 mL, 4.3 mmol) and 1.0 M tetrabutylammonium fluoride intetrahydrofuran (2.2 mL, 2.2 mmol). The mixture was stirred for 30 min.,evaporated, triturated with 1:10 mixture of CHCl₂/ether (50 mL), andstored at −20° C. for 30 min. After removal of the filtrate, the solidwas dissolved into a 1/1 mixture of CH₃CN/acetic acid (26 mL) and cooledto 0° C. Triacetoxyborohydride (0.367 g, 1.74 mmol) was added and themixture stirred for 30 min., evaporated, and evaporated twice fromtoluene. Silica gel chromatography 10-15% CH₃OH (1% conc. NH₄OH)/CH₂Cl₂yielded 0.212 g (0.89 mmol, 61%) of product.

Example 2 3,6-Dimethyl-5iodo-2-pyridone

2-Amino-3,6-dimethyl-5-iodopyridine (4.01 g, 16.2 mmol) was dissolved in0.5 M H₂SO₄ (70 mL) and the resulting solution cooled to 0° C. withstirring. A solution of sodium nitrite (3.3 g, 48.0 mmol) in H₂O (5 mL)was added dropwise over 10 min. at 0° C. with vigorous stirring. After1.5 h the cooling bath was removed and the mixture warmed to r.t. over 1h. The solid was vacuum filtered, washed with H₂O (50 mL), the solidsair dried and then dried under vacuum for 18 h, yielding 3.9 g (15.7mmol, 97%) of white solid.

Example 3 6-Amino-2,5-lutidine

6-Amino-2,5-lutidine (aminolutidine) was prepared by the method of Rao,K. V.; Venkateswarlu, P.; J. Heterocyclic Chem. (1975) 12:731-735, inparticular page 732, column 2, paragraph 2, titled“6-amino-2,5-lutidine.”

Example 4 2-Amino-3,6dimethyl-5-iodo Pyridine

7.0 g (38.5 mmole) of aminolutidine acetate was dissolved into H₂O (100mL) and solid NaOH was added until the pH of the solution was >10. 20 gof NaCl was dissolved into this solution and the aminolutidine wasextracted into CH₂Cl₂ (2×100 mL), dried over Na₂SO₄ and evaporated to anoil. To this oil was added CH₂Cl₂/Et₂O (80 mL, 1/1), the mixture cooledto ˜5° C. and 6.5 g (40 mmole) of ICl in CH₂Cl₂ (40 mL) was addeddropwise over 10 min. After addition was complete Et₂O (40 mL) was addedand the mixture removed from the ice-bath. After stirring for 1 hr. Et₂O(40 mL) was added, the solid filtered, washed with Et₂O and thesupernatant evaporated. The product was dissolved into 1N HCl, washedwith CH₂Cl₂, the aqueous phase made basic with solid NaOH and theproduct isolated by filtration. Yield 3.2 g (13 mmole, 68%).

Example 51,4-Anhydro-2-deoxy-3-O-[(1,1-dimethylethyl)diphenylsilyl]-D-erythro-pent-1-enitol

1,4-Anhydro-2-deoxy-3-O-[(1,1-dimethylethyl)diphenylsilyl]-D-erythro-pent-1-enitolwas prepared by the method of Farr, R. N.; Daves, G. D., Jr.; JCarbohydrate Chem. (1990) 9(5):653-660, in particular, page 658,paragraph 1, titled“1,4-Anhydro-2-deoxy-3-O-[(1,1-dimethylethyl)diphenylsilyl]-D-erythro-pent-1-enitol.”

Example 6 4-(2′-Deoxy-β-D-ribofuranosyl)-3,6-dimethyl-2-pyridone

A. A mixture of palladium (II) acetate (0.200 g, 0.89 mmol),triphenylarsine (0.542 g, 1.77 mmol) and CH₃CN (20 mL) was stirred underan Argon atmosphere at room temperature. A solution of1,4-anhydro-2-deoxy-3-O-[(1,1-dimethylethyl)diphenylsilyl]-D-erythro-pent-1-enitol(3.13 g, 8.83 mmol) and tributylamine (3.16 mL, 13.2 mmol) in CH₃CN (30mL) was added under an Argon atmosphere followed by addition of solid3,6-dimethyl-5-iodo-2-pyridone (2.2 g, 8.83 mmol). The mixture wasstirred at 60° C. for 18 h and then evaporated. Silica gelchromatography (5-7.5% CH₃OH/CH₂Cl₂) yielded 1.30 g (2.74 mmol, 31%) ofsolid.

B. The solid of Example 6A was dissolved in THF (40 mL) and cooled to 0°C. Acetic acid (0.19 mL) was added followed by 1 M tetrabutylammoniumfluoride (3.0 mL, 3.0 mmol). The mixture was stirred for 30 min.,evaporated, triturated with ether (60 mL) and the filtrate removed. Thesolid was dissolved in 1/1 CH₃CN/acetic acid (40 mL) and cooled to 0° C.Triacetoxyborohydride (0.876 g, 4.1 mmol) was added and the mixturestirred for 1 h at 0° C., evaporated and silica gel chromatography(10-12% CH₃OH/CH₂Cl₂) yielded 0.600 g (2.51 mmol, 92%) of product.

Example 7 Footprint Analysis.

DNase footprint analysis of a 370-bp restriction fragment containing thetarget sequence was preformed in the conventional manner (Froehler, B.C. and Ricca, D. J., J. Am. Chem. Soc. (1992) 114:8320-8322; Cooney, M.;Czernuszewicz, G.; Postel, E. H.; Flint, S. J.; Hogan, M. E., Science(1988) 241:456-459; Francois, J. C.; Saison-Benmoaras, T.; Helene, C;Nucleic Acids Res. (1988) 16:11431-11440; Matteucci, M.; Lin, K.-Y.;Butcher, S.; Moulds, C.; J. Am. Chem. Soc. (1991) 113:7767-7768). TheResults are shown below in Table 10. The triple-helix formation wasassessed via footprint assay for the analog targeted to both Select I(5′TCTCCCTCTCTTTTT3′)<Seq. ID No. 1> and Select II(5′TCTCTCTCTCTTTTT3′)<Seq. ID No. 2> cassettes vs. the T/5meC control(5meC is 5-methyl C). The results are at least 10-fold enhancement inbinding at pH =7.2. Specificity of binding was maintained.

TABLE 10 Cas I (μM) Cas II (μM) Sequence 10 1 0.1 10 1 0.1 5′TCTCTCTCTCTTTTT 3′ − − − + + − <Seq. ID No. 3> 5′ TNTNTNTNTNTTTTT 3′ − −− + + + <Seq. ID No. 4> 5′ UNUNUNUNUNUUUUU 3′ − − − + − − <Seq. ID No.5> 5′ TNTNNNTNTNTTTTT 3′ + + +/− − − − <Seq. ID No. 6> 3′TCTCTCTCTCTTTTT 5′ − − − + +/− − <Seq. ID No. 7> 3′ UNUNUNUNUNUUUUU 3′ −− − − − − <Seq. ID No. 8> + = protection − = no protection +/− = partialprotection N = 2-aminopyridine U = 2-pyridinone C = 5-methyl dC T =thymidine

Duplex Tm RNA DNA ODN Tm ΔTm Tm ΔTm Control 61.5 — 52.5 —2-aminopyridine NT — NT — 2-pyridinone 59.0 −2.5 47.0 −5.5 control = 5′TCTCTCTCTCTTTTT 3′ <Seq. ID No.3> 2-aminopyridine = 5′TPTPTPTPTPTPTPTPTTTTT 3′ <Seq. ID No. 4> 2-pyridinone = 5′TCTCTCTCTCUUUUU 3′ <Seq. ID No. 9> NT = no transition

All U.S. patent citations above are hereby expressly incorporated byreference at the locations of their citation. Specifically citedsections or pages of the above cited works are incorporated by referencewith specificity. The invention has been described in detail sufficientto allow one of ordinary skill in the art to make and use the subjectmatter of the following claims. It is apparent that certainmodifications of the methods and compositions of the following claimscan be made within the scope and spirit of the invention.

9 1 15 DNA Artificial Sequence Description of Artificial Sequence NovelSequence 1 tctccctctc ttttt 15 2 15 DNA Artificial Sequence Descriptionof Artificial Sequence Novel Sequence 2 tctctctctc ttttt 15 3 15 DNAArtificial Sequence Description of Artificial Sequence Novel Sequence 3tntntntntn ttttt 15 4 15 DNA Artificial Sequence Description ofArtificial Sequence Novel Sequence 4 tntntntntn ttttt 15 5 15 RNAArtificial Sequence Description of Artificial Sequence Novel Sequence 5nnnnnnnnnn nnnnn 15 6 15 DNA Artificial Sequence Description ofArtificial Sequence Novel Sequence 6 tntnnntntn ttttt 15 7 15 DNAArtificial Sequence Description of Artificial Sequence Novel Sequence 7tntntntntn ttttt 15 8 15 RNA Artificial Sequence Description ofArtificial Sequence Novel Sequence 8 nnnnnnnnnn nnnnn 15 9 15 DNAArtificial Sequence Description of Combined DNA/RNA Molecule DNA/RNAMixed Oligomer 9 tctctctctc uuuuu 15

What is claimed is:
 1. A compound of the formula:

wherein: each R¹ are independently H or a hydroxy protecting group, orboth R¹ groups are taken together to form a cyclic hydroxy protectinggroup; R² is H, F, —OR¹, or —OR⁶; R³ is H or —CH₃; each R⁴ of formula Iand II are independently H or an amine protecting group, or both R⁴groups of formula I are taken together to form a cyclic amine protectinggroup; R⁵ is H, —CH₃ or —C≡C —CH₃; and R⁶ is

and salts, solvates, resolved enantiomers and purified diastereomersthereof.
 2. The compound of claim 1 wherein the compound is of theformula:


3. The compound of claim 1 wherein R³ is —CH₃.
 4. The compound of claim1 wherein the compound is of formula I.
 5. The compound of claim 4wherein the compound is of the formula:


6. The compound of claim 5 with the proviso that the compound is not offormula III wherein each of R¹, R², R³, R⁴, and R⁵ are H.
 7. Thecompound of claim 5 wherein R³ is —CH₃.
 8. The compound of claim 7wherein the compound is of the formula:


9. The compound of claim 8 wherein: R² is H and R⁵ is H; R² is H and R⁵is —CH₃; R² is H and R⁵ is —C≡C—CH₃; R² is —OH and R⁵ is H; R² is —OHand R⁵ is —CH₃; or R² is —OH and R⁵ is —C≡C—CH₃.
 10. The compound ofclaim 9 wherein R² is H and R⁵ is —CH₃.
 11. An oligomer compoundcomprising a multiplicity of nucleosides linked by internucleosidelinkages wherein at least one nucleoside is a modified nucleosidecomprising a compound of the formula:

wherein: each R¹ are independently H or a hydroxy protecting group, orboth R¹ groups are taken together to form a cyclic hydroxy protectinggroup; R² is H, F, —OR¹, or —OR⁶; R³ is H or —CH₃; each R⁴ of formula Iand II are independently H or an amine protecting group, or both R⁴groups of formula I are taken together to form a cyclic amine protectinggroup; R⁵ is H, —CH₃ or —C≡C —CH₃; and R⁶ is

and salts, solvates, resolved enantiomers and purified diastereomersthereof.
 12. A method of detecting the presence, absence or amount of aparticular DNA duplex in a sample suspected of containing DNA comprisingcontacting the sample with an oligomer of claim 11 under conditionswherein a triple helix is formed between the oligomer and the particularDNA duplex.
 13. The oligomer compound according to claim 11, wherein atleast one of said internucleoside linkages is a phosphorothioatelinkage.